缺血预处理对L-6TG大鼠肌母细胞缺血再灌注中细胞凋亡的影响

目的:探讨缺血预处理对L-6TG大鼠肌母细胞缺血再灌注中细胞凋亡的影响。方法:将培养的L-6TG大鼠肌母细胞随机分为3组:①正常对照组(C组),②缺血再灌注组(IR组),③缺血预处理组(IP组),观测了培养上清中LDH、细胞内SOD、XOD、Ca2+含量的变化;采用MTT法检测线粒体的功能;利用流式细胞仪和细胞DNA电泳结果检测细胞凋亡情况;采用免疫组织化学的方法检测bcl-2及Caspase-3的蛋白表达情况,结合自动图像分析系统对其结果进行定量分析;在光镜下观察细胞的形态学改变。结果:缺血预处理可显著降低L 6TG大鼠肌母细胞IR 4h后培养上清中LDH、细胞内XOD、Ca2+含量及凋亡细胞百分率,增加细胞内SOD活性及线粒体呼吸功能,DNA电泳无梯状条带出现,bcl-2及Caspase-3的表达明显下调,细胞损伤明显减轻。结论:缺血预处理可明显减轻L-6TG大鼠肌母细胞缺血再灌注后的细胞损伤和细胞凋亡,其机制可能与减轻氧化损伤、调节细胞内钙稳态、减轻线粒体损伤、减少Caspase-3表达有关。

Effects of ischemic preconditioning on apoptosis during ischemia reperfusion in L-6TG rat skeletal myoblasts

Objective: To study the effects of ischemic preconditioning on apoptosis during ischemia reperfusion in L-6TG rat skeletal myoblasts Methods: Cultured L-6TG cells were divided into 3 groups:control group(C),ischemia-reperfusion group(IR),ischemic preconditioning group(IP).LDH in culture fluid,SOD,XOD,free calcium in L-6TG cell and mitochchondral respiration were evaluated in each group;apoptosis was detected by flow cytometer with PI staining method and agarose gel electrophoresis;the immuneohistochemical method was used to determine the expression of bcl-2 and Caspase-3;the morphoiogic changes were observed with microscope.Results: Compared with IR group,in IP group,LDH in culture fluid,XOD,free Calcium in L-6TG cell and apoptotic percentage all decreased significantly,while SOD in L-6TG cell and mitochchondral respiration increased;DNA fragmentation analysis of L-6TG cell showed no laddering pattern;the expression of bcl-2 and Caspase-3 were downregulated significantly;structural change of L-6TG cell was not obvious.Conclusion: IP can lessen ischemia reperfusion injury and apoptosis through lessening oxidative injury and mitochchondral injury and adjusting calcium dyshomeostasis,overexpression of bcl-2 and downexpression of Caspase-3.